CN113234601B - Novel marine fungus strain and application thereof in drought stress resistance of plants - Google Patents
Novel marine fungus strain and application thereof in drought stress resistance of plants Download PDFInfo
- Publication number
- CN113234601B CN113234601B CN202110356838.4A CN202110356838A CN113234601B CN 113234601 B CN113234601 B CN 113234601B CN 202110356838 A CN202110356838 A CN 202110356838A CN 113234601 B CN113234601 B CN 113234601B
- Authority
- CN
- China
- Prior art keywords
- strain
- westerdykella
- plants
- marine fungus
- cgmcc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Soil Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention aims to provide a new marine fungus strain Westerdykella sp.CGMCC No.20219 with the number of P201, which is separated from the circular leaf salt claw in Xinjiang and arid desert regions, and an application thereof in plant drought stress, wherein the strain grows well under the condition of 0-8% NaCl at 20-30 ℃ and pH of 6.0-7.0 and 0-1% NaCl. The strain has the advantages of improving the content of plant catalase, promoting the drought resistance of plants, being applicable to enhancing the drought resistance of plants and having good application prospect of being developed into biological fertilizers.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a novel marine fungus strain and the technical field of application of the novel marine fungus strain in drought resistance of plants.
Background
Drought is one of the most common abiotic stresses, is one of the main limiting factors of grain safety and plant productivity worldwide, has adverse effects on the growth, development, agronomic yield and the like of crops, and causes land degradation, and the damage degree is equivalent to the sum of losses caused by other natural disasters, wherein the losses account for about 53% of the influence of natural disasters on the crops. The inter-government climate change committee (IPCC) reports that the frequency of drought will increase at the end of this century as climate changes. And corn and wheat are main crops in drought frequent areas, so that the improvement of stress resistance of the corn and the wheat under drought stress is of great significance.
Drought stress causes plants to produce negative effects, such as oxidative stress of plants caused by ionic stress and osmotic stress, resulting in the production of reactive oxygen species (reactive oxygen species, ROS) that are detrimental to plants. Osmotic stress leads to a general decrease in seed germination rate. The plant leaves are curled and yellowing, plant height, root length and fresh dry weight are gradually reduced, the pore density of the leaves is obviously increased, and the pore air permeability and water potential are gradually reduced. In addition, drought also has significant adverse effects on physiological biochemistry, metabolism and the like of plants, such as higher chlorophyll content of plant leaves under drought stress in plants with higher tolerance and significantly reduced chlorophyll content in plants with poor tolerance; the root system activity of the seedling is enhanced, the antioxidant enzyme activity is improved, and the levels of abscisic acid (ABA), proline (PRO), soluble sugar and Polyamine (PAS) are obviously increased under drought stress.
Endophytic fungi are fungi that live inside plant tissue without causing significant damage to the host during all or some of their life history. Endophytic fungi widely exist in different regions and different plant tissues, have rich diversity and remarkable ecological functions, and are important components of global biodiversity. In the long-term evolution of the ecosystem, endophytic fungi form symbiotic relations with various plants. The endophytic fungi and the host mutually select, mutually adapt and co-evolve, and the community structure of the endophytic fungi shows host preference and tissue preference. Endophytic fungi can improve plant growth conditions and improve the capability of resisting environmental stresses such as saline alkali, drought, plant diseases and insect pests and the like.
Researches show that the symbiosis of the lichen fungi and mycorrhizal fungi and plants can improve the drought resistance of hosts, but the researches on improving the drought resistance of plants by the endophyte are relatively few, and the researches are mainly focused on a few grass endophyte. Research results show that the endophytic fungi of grass can improve drought resistance of host plants and react to drought through different mechanisms such as escape (avoidance), tolerance (tolerance) and rapid recovery (recovery). To address and mitigate the hazards caused by drought, endophytic fungi can adjust plant physiological and morphological characteristics. In plot and field experiments, researchers find that endophytic fungi can obviously promote drought tolerance of hosts such as festuca arundinacea, nigella sativa, ryegrass and the like; compared with the uninfected plants, the drought resistance indexes of the plants infected by the endophytic fungi, such as seed germination capacity, leaf growth speed, leaf area and the like, leaf curling capacity, root system biomass, root system depth and CO 2 The exchange rate is more excellent, and the death rate of plants under drought conditions is lower. In addition to morphological differences, plants infected with endophytes differ in physiological and biochemical aspects from plants infected with endophytes, and the improvement in drought resistance of plants infected with endophytes may be related to the improvement in the levels of hormones in plants, such as ABA, polyamines, osmotically active factors, such as SOD, POD, etc., protective enzyme systems, alkaloids, such as line alloys, ergopeptine alkaloids, etc.
The invention screens a marine fungus belonging to the genus Westerdykella from plant circular leaf salt claw (Kalidiumschrenkianum) in arid desert area of Xinjiang, and determines the fungus and Westerdykella centenaria CBS142400 through molecular identification and systematic identification of the strain T The homology is 97.42%, and the strain is a new strain of the genus. The application of the genus strain in agriculture has not been reported before. The strain screened by the invention has the capability of promoting drought resistance of plants and has wide application prospect in the aspect of improving stress resistance of plants.
The invention comprises the following steps:
the invention aims to provide a new strain P201CGMCC No.20219 in the Westerdykela genus, and the microbial fertilizer of the strain is prepared by utilizing a liquid fermentation technology, so that the content of plant catalase can be remarkably improved, and the drought resistance of plants is promoted.
The invention aims at realizing the following technical scheme:
the invention aims to provide a new marine fungus strain Westerdykella sp.P201CGMCC No.20219 separated from the circular leaf salt claw in Xinjiang and arid desert regions and application thereof, and the strain has the advantages of improving the content of plant catalase and the drought resistance of plants, can be applied to enhancing the drought resistance of plants, and has good application prospect of being developed into biofertilizer.
The invention firstly provides a novel strain westerdykela sp.P201 in marine fungus, which is preserved in the national institute of microbiological culture preservation of China general microbiological culture Collection center of China national academy of sciences of China No. 3 of the national academy of sciences of China, including North Chen West Lu No. 1 in the Korean area of Beijing, 7 months and 6 days in 2020, with a preservation number of CGMCC No.20219. The sequence of the LSU rDNA D1/D2 region is shown as SEQ ID NO. 1.
The culture medium of the new strain Westerdykela sp.P201CGMCC No.20219 with the number of P201 in the marine fungus genus provided by the invention: 30g/L of yeast extract powder, 3g/L of soybean peptone and 16g/L of agar powder, wherein the colony is round when grown on a culture medium for 12 days, the diameter is 2.2-2.5cm, the front surface is mussel white, the back surface is chestnut brown, no secretion exists, the short velvet is shaped, black microspores can be observed occasionally, and the mycelia have endophytes. The strain grows under the condition of 0-8% NaCl, and grows well under the conditions of 20-30 ℃ and pH of 6.0-7.0 and 0-1% NaCl.
Amplifying the sequence of the LSU rDNA D1/D2 region of the new strain Westerdykella sp.P201CGMCC No.20219 strain with the number of P201 strain marine fungus as a template, performing DNA sequencing to obtain the sequence of the LSU rDNA D1/D2 region with the length of 596bp (see a sequence table in the attached list), performing homology comparison analysis on the sequence of the LSU rDNAD1/D2 of the new strain Westerdykella sp.CGMCC No.20219 strain with the sequence of a representative strain recorded in GenBank database, and obtaining the result of the homology comparison with the standard mode strain Westerdykella centenaria CBS142400 of the genus T Homology of 97.42% is a potential new species of this genus, and at present, the strain has not been formally named in microbiological taxonomy, and the tentatively named P201 strain is named Westerdykela sp.
The strain Westerdykela sp with the number of P201 has salt tolerance, and can improve drought stress resistance of plants, and improve peroxidase content of plants, so as to improve oxidation resistance of plants and relieve influence of environmental stress on plants. Thereby playing a very important role in preventing and controlling plant diseases.
Furthermore, the invention also provides a novel bacterial Westerdykela sp.P201 bacterial agent of marine fungi, which is characterized by being prepared by the following steps:
(1) Inoculating new strain Westerdykela sp.P201CGMCC No.20219 of marine fungus on a solid culture medium prepared by 30g/L yeast extract powder, 3g/L soybean peptone and 16g/L agar powder for activation;
(2) Transferring the strain activated in the step (1) into 100mL of Charles liquid culture medium at 28 ℃ and culturing at 180rpm for 48 hours to obtain seed liquid;
(3) Inoculating the seed solution prepared in the step (2) into a Charles liquid culture medium for fermentation at a ratio of 2%, and culturing at 28 ℃ for 96 hours at 200 rpm;
(4) Diluting the bacterial liquid obtained in the step (3) with sterilized water to obtain a total concentration of 1×10 viable bacteria 8 -1×10 10 cfu/mL of a new strain Westerdykela sp.P201 microbial inoculum of the genus marine fungus.
The invention has the following beneficial technical effects:
(1) The invention screens a marine fungus belonging to the genus Westerdykella from the plant circular leaf salt claw (Kalidium schrenkianum) in the arid desert region of Xinjiang, and determines the number of the strain P201, westerdykella sp.CGMCC No.20219 and Westerdykella centenaria CBS142400 through molecular identification and systematic identification of the strain T The homology is 97.42%, and the strain is a new strain of the genus. The application of the genus strain in agriculture is recently reported. The strain screened by the invention has the capability of promoting drought resistance of plants and has wide application prospect in the aspect of improving stress resistance of plants. The strain can be used as a biological agent, not only improves the stress resistance of plants and reduces the dosage of chemical fertilizers, but also can save the expenditure of farmers, increase the economic income and have good ecological benefit and social benefit.
(2) The new strain in the marine fungus genus provided by the invention adopts the strain with the number of P201, namely Westerdykela sp.CGMCC No.20219, can resist the high-salt drought environment, and can enhance the resistance of plants to environmental stress. After the new marine fungus strain Westerdykela sp.P201CGMCC No.20219 provided by the invention is adopted for treatment, the plant height of wheat is improved by 10.15%, the root length is improved by 13%, and the average weight of a single plant is improved by 7.14%.
(3) The novel marine fungus strain Westerdykella sp.P201CGMCC No.20219 provided by the invention can also obviously improve the contents of plant peroxidase, coenzyme A ligase and polyphenol oxidase, and can be used as a wide probiotic to be applied to other plants.
Description of the drawings:
FIG. 1 is a diagram showing the morphology of the front and back sides of a colony of the strain Westerdykela sp.CGMCC No.20219 with the number of P201.
Wherein the graph A is a reverse side shape graph; fig. B is a front view.
FIG. 2 is a diagram showing the microscopic morphology of mycelia and spores of the strain Westerdykela sp.CGMCC No.20219 with the number of P201.
Wherein figure a is a mycelium microscopic morphology; panel B is a spore photomicrograph.
FIG. 3 is a phylogenetic tree diagram of the strain Westerdykela sp.CGMCC No.20219 with the number P201.
FIG. 4 is a diagram of the wheat peroxidase production by the strain Westerdykela sp.CGMCC No.20219 with the number of P201.
FIG. 5 is a diagram of the coenzyme A ligase produced by wheat under the condition that the number of the bacterial strain P201 is Westerdykella sp.CGMCC No.20219.
FIG. 6 is a diagram of the wheat producing polyphenol oxidase by the westerdykela sp.CGMCC No.20219 strain with the number of P201.
The specific embodiment is as follows:
the following are specific examples of the present invention, but the present invention is not limited thereto.
Charles medium (g/L): naNO 3 2，K 2 HPO 4 1，KCl 0.5，MgSO 4 0.5，FeSO 4 0.01, sucrose 30, agar powder 16.LB medium: tryptone 10, yeast extract 5, naCl 10.
Main instruments and equipment: PL202 electronic balance, meltler-tolido instruments limited; HPX-9272 digital display electric heating incubator, shanghai Boqing Utility company medical equipment factory; an LDZX-40BI autoclave, shanghai Shen An medical equipment factory; GZX-9420 electrothermal constant temperature blast drying oven, shanghai BoXie Co., ltd; SW-CJ-2F ultra clean bench, shanghai BoXie Co., ltd; fcycler 96-well PCR reactor, bio-Rad company; DYY-6C electrophoresis apparatus, beijing Liuyi instrument factory; GEL imaging analyzer, bio-Rad, GEL Doc 2000; an LDZX-50KB vertical pressure steam sterilizer, shanghai Shen An medical equipment factory; biolog, biotek instruments, usa; vernier calipers, shanghai work volume with company Limited; WHB 96 micro-well plate, shanghai City Uygur pal Biotechnology Co., ltd; a 1000ml pipette, shanghai Ai Bende Biotechnology International trade Co., ltd; C21-RT2103 electromagnetic oven, EG720FF1-NR microwave oven, guangdong American household electrical appliance manufacturing Co., ltd; BL3100 electronic balance, sartorius, germany; PHS-3B pH meter, shanghai Red beneficial instruments and meters Co., ltd; biolog microbiological automation system, biolog company, usa.
All strains and raw materials used in the invention, as well as the culture conditions and methods of the selected strains are well known in the art, and all the raw materials involved in the invention are commercial products.
Embodiment one: the separation, the screening, the purification and the preservation of the strain Westerdykela sp.CGMCC No.20219 with the number of P201 strains
Screening strains: the strain Westerdykela sp.CGMCC No.20219 is separated from the circular leaf salt paw in Xinjiang and arid desert region. Washing plant tissue with tap water, sequentially soaking in 75% alcohol for 1min,3.3% sodium hypochlorite for 5min, and 75% alcohol for 0.5min, washing with sterile water for 3 times, and sucking with filter paper. Under aseptic condition, weighing 1.0g of aerial parts and root parts of plants respectively, adding into 50mL sterilized normal saline (0.85%), pulverizing with pulverizer, mixing, diluting with standard gradient, and diluting with sterile water to obtain 10 -2 -10 -5 A diluent at a concentration. 100. Mu.L of each concentration of the dilution was spread on a solid medium plate by a conventional spreading method, and the plate was placed in a constant temperature incubator at 30℃for cultivation. After bacterial colonies grow out on the plate for 48 hours, single colonies on the plate are picked for purification culture. And transferring the purified single colony to a culture medium inclined plane for preservation for later use.
Culture medium: 30g/L yeast extract powder, 3g/L soybean peptone and 16g/L agar powder.
Embodiment two: identification of the Westerdykela sp.CGMCC No.20219 Strain with the number of P201
1. Morphological observations of strains
The strain with the number of P201 is Westerdykela sp.CGMCC No.20219, can grow on MEA and PDA culture medium, normally grows at 20-30 ℃, has the optimal culture temperature of 28 ℃ and can resist 8% NaCl. After culturing for 12 days in MEA medium at 28deg.C, the colony is round and 2.2-2.5cm in diameter, the front surface is mussel white, the back surface is chestnut brown, no secretion is generated, and black microspores and endophyte can be observed occasionally, see figure 1. Hyphal and spore morphology was observed under a microscope, see fig. 2. Conidium device is spherical, black, scattered, surface-grown, has mastoid-shaped opening, and has a diameter of 100-200 μm. The conidium has thin wall, dark brown and angular texture. The conidiophore is simple and is isolated. Spore-forming cells are colorless or slightly pale brown, ampoule-shaped, and bud on the inner wall. The spore is spherical, nearly spherical, to egg-shaped, light brown, thin-walled, single-cell and no diaphragm, and oil drops are more visible in the spore, and the spore size is 3-5.5 (4.3+/-0.73) multiplied by 2.5-5 (4.1+/-0.82) mu m. Available on Biolog FF plate: tween 80, ribitol, D-arabinose, D-arabitol, a-cyclodextrin, erythritol, gentiobiose, maltitol, a-methyl-D-glucoside, D-ribose, salicin, D-sorbitol, D-xylose, β -hydroxybutyric acid, succinic acid, L-asparagine, ornithine, 2-aminoethanol, humic acid. As can be seen by comparing with the common strains in the genus of the marine fungi, the strain with the number of P201 is obtained by screening in the application, and the strain is Westerdykela sp.CGMCC No.20219, which has the vivid characteristics and properties of the new strains in the marine fungi and the representativeness of the new strains in the genus of the marine fungi.
PCR amplification of the D1/D2 region of LSU rDNA
The strain with the number of P201 strain Westerdykella sp.CGMCC No.20219 is inoculated in a liquid Charles culture medium, cultured for 4d at the constant temperature of 28 ℃, centrifugally collected, and washed 3 times by TE. After the thalli are ground by liquid nitrogen, DNA extraction refers to the method of Gerrits and the like, DNA is naturally dried after being washed by 70 percent ethanol, 50 mu L of sterile double distilled water is added, and the mixture is dissolved for 2 hours at 4 ℃ and preserved at 20 ℃ for standby. The PCR amplification of the LSU rDNA D1/D2 region uses fungal universal primers:
NL1：5′-GCATATCGGTAAGCGGAGGAAAAG-3′
NL4：5′-GGTCCGTGTTTCAAGACGG-3′；
the amplification conditions were: 95 ℃ for 5min;94 ℃ 1min,52 ℃ 1min and 72 ℃ 2min for 35 cycles; and at 72℃for 10min. And (3) purifying and recycling the PCR product by cutting gel, adding deionized water for dissolving, sequencing, and enabling the sequence of the strain to be see a gene sequence table.
3. Construction and analysis of phylogenetic tree
BLAST comparison of the sequence of the LSU rDNA D1/D2 region obtained from the experimental strain with known sequences in the GenBank database is performed to determine the species closest to the relatedness of the experimental strain. The sequences of related species are obtained from a database, and combined with the standard model fungus sequences of a fungus biodiversity research center database (http:// www.cbs.knaw.nl/databases /), the MEGA7.0 software package is used for carrying out cluster analysis and phylogenetic tree construction by adopting a common adjacent method (Neighbor-training method) to determine the biological classification status of the strains.
4. Molecular characterization of strains
In order to further determine the classification status of the strain Westerdykela sp.CGMCC No.20219 with the number of P201, the corresponding model strain sequence is prepared by comparing and analyzing the strain LSU region sequence, and the MEGA7.0 is utilized for constructing and analyzing a phylogenetic tree, as shown in the attached figure 3. The result shows that the experimental strain with the number of P201 strain Westerdykella sp.CGMCC No.20219 is Westerdykella genus and Westerdykella centenaria CBS142400 T In one branch, the support rate was 71% and the maximum homology was 97.42%. Homology with other standard mode bacteria of the genus is lower than 97.0%, the official microorganism taxonomic naming of the strain with the number of P201 strain Westerdykella sp.CGMCC No.20219 is not carried out at present, the tentative strain name is Westerdykella sp.P201, the molecular level of the strain with the number of P201 strain Westerdykella sp.CGMCC No.20219 is identified by the molecular level, and the strain with the number of P201 strain Westerdykella sp.CGMCC No.20219 is a new strain in the marine fungus Westerdykella sp.according to the general knowledge of fungus identification in the microorganism field.
At present, the strain P201 is Westerdykela sp.CGMCC No.20219 which is preserved in the Budapest treaty microorganism International deposit unit before the application date: china general microbiological culture Collection center (CGMCC). Address: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101, the preservation date is 7 months and 6 days in 2020, and the preservation number is CGMCC No.20219.
Embodiment III: preparation of bacterial agent with P201 strain Westerdykela sp.CGMCC No.20219
Inoculating westerdykela sp.CGMCC No.20219 with the number of P201 strain on a solid culture medium prepared by 30g/L yeast extract powder, 3g/L soybean peptone and 16g/L agar powder, activating, transferring into 100mL Charles liquid culture medium (500 mL triangular flask with small amount of glass beads), shake culturing at 28deg.C and 180rpm for 48h, obtaining seed liquid. Seed solution was inoculated in 2% ratio to Charles liquid medium (500 mL flask with a small amount of glass beads) and fermented, at 28℃and 200rpm for 96 hours. Filtering the fermentation broth with double-layer gauze to remove larger fungus balls and mycelia, and diluting with sterilized water to obtain a fungus solution with concentration of 1×10 6 -1×10 8 cfu/mL of microbial inoculum.
Embodiment four: drought stress resistance of wheat under the condition of P201 strain Westerdykella sp.CGMCC No.20219
Based on the technical scheme described in the third embodiment, wheat seeds with full grains and no obvious damage are selected, sterilized for 5min by using 0.1% mercuric chloride, washed by sterile water, dried in the air and immersed in 1/100 concentration bacteria solution for 4h at room temperature. After the control treatment was sterilized by the same method, the seeds were immersed in sterilized distilled water. The wheat seeds of the treatment group and the control group are respectively planted into hole planting pots, each hole is 4 grains, the depth is about 1.5cm, and the wheat seeds are placed in a climatic chamber for cultivation. Culturing at 20deg.C for 8 hr without light; light is irradiated for 2 hours at the temperature of 22 ℃ and 30%; 25 ℃ and 100% illumination for 12 hours; 30% light is illuminated for 2h. And water is poured once a day, and the watering amount is controlled to be consistent. After germination, the seedlings were irrigated every 5 days (about 10mL per hole). And (3) normal water management, namely, keeping the relative water content (soil relative water content, SRWC, accounting for the percentage of the water holding capacity of the soil) of 70-75% by adopting a weighing method. Drought tests were performed after 30d germination. In the experiment, moderate drought stress is selected for drought treatment, namely SRWC is 40% -45%, and a control group with normal water content is arranged at the same time. After the natural drought, the soil moisture content is controlled by adopting a weighing method, and the moisture is supplemented to the drought stress level about 18:00 pm every day. The test was ended 10 days after drought treatment. Sampling about 7:30 a.m. the next morning, and measuring the growth index and physiological index. The results show that wheat treated with the westerdykela sp.cgmcc No.20219 microbial inoculum, which is a strain P201, shows stronger advantages in seedling height and root length than the control group, although the germination rate is reduced, and the individual weight is increased. The method has the advantages that the treatment of the strain Westerdykella sp.CGMCC No.20219 with the number of P201 has a certain promotion effect on drought stress resistance of wheat.
Table 1: growth index of wheat treated by P201 bacterial liquid under drought stress
| Numbering device | Height of plant (cm) | Root length (cm) | Weight (g) | Number of sprouting | Average weight of individual plants (g) |
| CK | 19.7 | 10 | 15.98 | 38 | 0.42 |
| P201 | 21.7 | 11.3 | 14.25 | 32 | 0.45 |
Note that: the tables are all average values
Fifth embodiment: promoting effect of P201 strain Westerdykela sp.CGMCC No.20219 on peroxidase production of wheat
POD (EC 1.11.1.7) is widely used in animals, plants, microorganisms and cultured cells, can catalyze hydrogen peroxide to oxidize phenols and amine compounds, and has the dual function of eliminating toxicity of hydrogen peroxide and phenols and amines.
Based on the description of the fourth embodiment, the plant sample is treated: about 0.1g of leaf tissue was weighed, and 1mL of the extract was added thereto to perform ice bath homogenization. And (5) centrifuging at the temperature of 8000g for 10min at 4 ℃, taking supernatant, and placing on ice for testing.
According to the instructions of a Peroxidase (POD) kit, 10. Mu.L of a sample and 190. Mu.L of a working solution were added to a 96-well plate, mixed well, and absorbance A1 at 470nm for 1min and absorbance A2 after 2min were recorded. Delta a=a2-A1 is calculated.
POD activity calculation:
the calculation formula for the 96-well plate assay is as follows:
POD (U/g fresh weight) =Δa×v inverse total (w×v samples ++v samples total)/(0.005 ++t=4000×Δa ++w)
As shown in FIG. 4, the peroxidase content in the wheat leaves treated with the bacterial liquid of the strain P201, westerdykela sp.CGMCC No.20219, reached 194.27U/g, whereas the control group had only 139.68U/g, which is very different. It was demonstrated that strain P201 has a promoting effect on wheat peroxidase production under drought stress.
Example six: promoting action of P201 strain Westerdykela sp.CGMCC No.20219 on coenzyme A ligase and polyphenol oxidase produced by wheat
4-coumaric acid: coenzyme A ligase (4-coumarate: coAliase, 4 CL) is a key enzyme for connecting phenylpropionic acid pathway and lignin specific synthesis pathway, and is mainly used for catalyzing cinnamic acid to generate corresponding cinnamic acid coenzyme A ester, and is a metabolic flow direction regulating point for synthesizing lignin and other phenylpropanes. The enzyme is mainly present in higher plants, yeasts and fungi.
The polyphenol oxidase PPO (EC 1.10.3.1) mainly exists in animals, plants, microorganisms and cultured cells, is a copper-containing oxidase, can oxidize monophenols and dihydric phenols to generate quinones, and thus causes browning, and is closely related to fruit and vegetable processing, tea quality, tissue culture and the like. In plant tissue, PPO is bound to the inner capsule membrane and is inactive in its natural state, but PPO is activated after tissue homogenization or injury, thus exhibiting activity. The lignin and the quinone compounds are catalyzed to form a protective shield to prevent cells from being damaged, and the quinone compounds can directly play a role in stress resistance. The sample treatment method is the same as in example five.
According to the 4-coumaric acid kit, 10. Mu.L of the sample and 190. Mu.L of the working solution were added to a 96-well plate, mixed well, and reacted at 40℃for 30 minutes at 333nm to record absorbance A1 (control tube) and absorbance A2 (test tube). Delta a=a2-A1 is calculated.
4CL activity calculation:
the calculation formula for the 96-well plate assay is as follows:
4CL (nmol/min/g fresh weight) = [ ΔA×V inverse total ≡ε×d ] ×10 9 ](w×v samples/V samples total)/(t=63.49×Δa/W)
Epsilon: 4-coumarate-CoA molar extinction coefficient, 2.1X10 4 L/mol/cm；
d: 96-well plate optical path, 0.5cm
According to the PPO kit, the treated sample is placed in a water bath at 25 ℃ for 10min, cooled to room temperature in a water bath at 95 ℃ for 5min, 10000g is centrifuged at 25 ℃ for 10min, the supernatant is collected, 200 mu L of the supernatant is taken into a 96-well plate, absorbance of a measuring tube and a control tube is detected at 525nm, and deltaA=A measurement-A control is calculated.
PPO activity calculation:
the calculation formula for the 96-well plate assay is as follows:
PPO (U/g fresh weight) =Δa×v inverse total (w×v samples ++v samples total) ≡0.005 ≡t=120×Δa ≡w)
The results are shown in the accompanying figures 5 and 6, the content of 4CL in the wheat leaves treated with the bacterial liquid with the number of P201 bacterial Westerdykela sp.CGMCC No.20219 reaches 640.6nmol/min/g, and the content of the control group is only 583.3nmol/min/g; the PPO content reaches 72.5U/g, and the control group content is only 65.1U/g, and although there is no significant difference between the treatment group and the control group, the increase of the yields of the two enzymes indicates that the strain P201 has a certain promotion effect on 4CL and PPO production of wheat under drought stress.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of the present invention, which is optimized without departing from the spirit of the design of the present invention.
Sequence listing
<110> institute of microbiological applications of the academy of agricultural sciences of Xinjiang (Xinjiang-Asian Biotechnology research and development center of China)
<120> a new marine fungus species and its application in drought stress resistance of plants
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 596
<212> DNA
<213> Westerdykella sp. P201
<400> 1
tccaaatagg aggaaaagaa accaacaggg attgccctag taacggcgag tgaagcggca 60
acagctcaaa tttgaaatct ggccctctca gggtccgagt tgtaatttgt agagggtgct 120
ttggcgttgg ctgtggtcta agttccttgg aacaggacgt cacagagggt gagaatcccg 180
tacgtggccg ccagcctccg ccgtgtaaag ccccttcgac gagtcgagtt gtttgggaat 240
gcagctctaa atgggaggta aattccttct aaagctaaat attggccaga gaccgatagc 300
gcacaagtag agtgatcgaa agatgaaaag cactttggaa agagagtcaa aaagcacgtg 360
aaattgttga aagggaagcg cttgcagcca gacttgcctg tagttgttca ccttggcttt 420
tgccttgggc attcttctac gggcaggcca gcatcagtcc ggacggttgg ataaatgctt 480
tctcaatgta cctctcttcg gggaggactt ataggggtta gcgacataca accagtctgg 540
actgaggtcc gcgcatctgc taggatgctg gcgtaatggc tgtaagcggc cgtgaa 596
Claims (4)
1. Marine fungusWesterdykellasp.P201, characterized in that said marine fungusWesterdykellaThe microorganism preservation number of sp.P201 is CGMCC No.20219.
2. Marine fungusWesterdykellaThe sp.P201 microbial inoculum is characterized by being prepared and obtained according to the following steps:
(1) Sea fungiWesterdykellaInoculating sp.P201CGMCC No.20219 on a solid culture medium prepared by 30g/L yeast extract powder, 3g/L soybean peptone and 16g/L agar powder for activation;
(2) Transferring the strain activated in the step (1) into 100mL of Charles liquid culture medium at 28 ℃ and culturing at 180rpm for 48 hours to obtain seed liquid;
(3) Inoculating the seed solution prepared in the step (2) into a Charles liquid culture medium for fermentation at a ratio of 2%, and culturing at 28 ℃ for 96 hours at 200 rpm;
(4) Filtering the fermentation liquor obtained in the step (3) by double-layer gauze to remove larger bacteria balls and mycelia, and diluting with sterilized water to obtain a total concentration of viable bacteria of 1×10 8 -1×10 10 Marine fungi of cfu/mLWesterdykella spP201 microbial inoculum.
3. The marine fungus of claim 1WesterdykellaUse of sp.p201 strain in enhancing the resistance of wheat to drought stress.
4. A marine fungus according to claim 3WesterdykellaThe application of sp.P201 strain in enhancing the drought stress resistance of wheat is characterized in that the novel marine fungus strainWesterdykellaP201 has the effect of promoting the production of peroxidase, coenzyme a ligase and polyphenol oxidase by wheat.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110356838.4A CN113234601B (en) | 2021-04-01 | 2021-04-01 | Novel marine fungus strain and application thereof in drought stress resistance of plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110356838.4A CN113234601B (en) | 2021-04-01 | 2021-04-01 | Novel marine fungus strain and application thereof in drought stress resistance of plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113234601A CN113234601A (en) | 2021-08-10 |
| CN113234601B true CN113234601B (en) | 2023-04-25 |
Family
ID=77130927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110356838.4A Active CN113234601B (en) | 2021-04-01 | 2021-04-01 | Novel marine fungus strain and application thereof in drought stress resistance of plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113234601B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061550B (en) * | 2021-04-01 | 2023-03-24 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Lactobacillus new strain Z6 and application thereof in food |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001078758A (en) * | 2000-08-23 | 2001-03-27 | Tokuyama Corp | Method for producing enzyme-containing solution |
| CN104762216A (en) * | 2014-12-26 | 2015-07-08 | 南京晓庄学院 | Fungus strain capable of resisting salt stress, and breeding method and application thereof |
| WO2018184817A1 (en) * | 2017-04-06 | 2018-10-11 | Novozymes A/S | Cleaning compositions and uses thereof |
| EP3478811A1 (en) * | 2017-04-06 | 2019-05-08 | Novozymes A/S | Cleaning compositions and uses thereof |
| CN110343620A (en) * | 2019-07-01 | 2019-10-18 | 福建农林大学 | One plant can Adsorption of Cadmium plant rhizosphere fungi F9 and its application |
| CN110582205A (en) * | 2017-03-01 | 2019-12-17 | 靛蓝股份公司 | Endophytic plant composition and method for improving plant traits |
| CN111040962A (en) * | 2019-11-29 | 2020-04-21 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | New strain marine bacterial strain XAAS-72 and application thereof in plant antibiosis and growth promotion |
| JP2020516245A (en) * | 2017-04-06 | 2020-06-11 | ノボザイムス アクティーゼルスカブ | Detergent composition and use thereof |
| CA3135069A1 (en) * | 2019-04-05 | 2020-10-08 | Novozymes A/S | Redox enzymes in animal feed compositions |
| CA3135056A1 (en) * | 2019-04-05 | 2020-10-08 | Novozymes A/S | Redox enzymes in animal feed compositions |
| CN114934002A (en) * | 2022-06-30 | 2022-08-23 | 塔里木大学 | Novel actinomycete species and application thereof in drought resistance and growth promotion of plants |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018535664A (en) * | 2015-10-07 | 2018-12-06 | ノボザイムス アクティーゼルスカブ | Polypeptide |
| EP3737737A4 (en) * | 2018-01-09 | 2021-10-06 | Novozymes A/S | Use of enzyme in removing airborne particulate matter from textile |
-
2021
- 2021-04-01 CN CN202110356838.4A patent/CN113234601B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001078758A (en) * | 2000-08-23 | 2001-03-27 | Tokuyama Corp | Method for producing enzyme-containing solution |
| CN104762216A (en) * | 2014-12-26 | 2015-07-08 | 南京晓庄学院 | Fungus strain capable of resisting salt stress, and breeding method and application thereof |
| CN110582205A (en) * | 2017-03-01 | 2019-12-17 | 靛蓝股份公司 | Endophytic plant composition and method for improving plant traits |
| WO2018184817A1 (en) * | 2017-04-06 | 2018-10-11 | Novozymes A/S | Cleaning compositions and uses thereof |
| EP3478811A1 (en) * | 2017-04-06 | 2019-05-08 | Novozymes A/S | Cleaning compositions and uses thereof |
| JP2020516245A (en) * | 2017-04-06 | 2020-06-11 | ノボザイムス アクティーゼルスカブ | Detergent composition and use thereof |
| CA3135069A1 (en) * | 2019-04-05 | 2020-10-08 | Novozymes A/S | Redox enzymes in animal feed compositions |
| CA3135056A1 (en) * | 2019-04-05 | 2020-10-08 | Novozymes A/S | Redox enzymes in animal feed compositions |
| CN110343620A (en) * | 2019-07-01 | 2019-10-18 | 福建农林大学 | One plant can Adsorption of Cadmium plant rhizosphere fungi F9 and its application |
| CN111040962A (en) * | 2019-11-29 | 2020-04-21 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | New strain marine bacterial strain XAAS-72 and application thereof in plant antibiosis and growth promotion |
| CN114934002A (en) * | 2022-06-30 | 2022-08-23 | 塔里木大学 | Novel actinomycete species and application thereof in drought resistance and growth promotion of plants |
Non-Patent Citations (9)
| Title |
|---|
| from TYPE material.《Genbank Database》.2018, * |
| NCBI.Westerdykella centenaria 28S rRNA gene, partial sequence * |
| 中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性(英文);王洁等;《微生物学通报》;20150220(第02期);全文 * |
| 海洋来源真菌生物活性物质研究进展;付逸群等;《山东化工》;20191123(第22期);全文 * |
| 海洋真菌Westerdykella dispersa FS350次级代谢产物的分离纯化与鉴定;许建林等;《广东药科大学学报》;20180410(第02期);全文 * |
| 海洋真菌Westerdykella dispersa和Phomopsis lithocarpus次级代谢产物及其生物活性研究;许建林;《中国优秀硕士学位论文全文数据库（电子期刊）》;20190115;全文 * |
| 海洋真菌来源的抗植物病原菌活性天然产物;王聪等;《中国海洋药物》;20181215(第06期);全文 * |
| 蔡永萍 等主编.实验五 环境胁迫对植物幼苗光合特性及相关生理指标的影响.《生物学综合实验教程》.中国农业大学出版社,2020,第32页. * |
| 辽宁省农田作物根围的真菌(III）;梁晨 等;《沈阳农业大学学报》;20030225;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113234601A (en) | 2021-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111876351B (en) | Bacillus belgii and application thereof in relieving apple continuous cropping obstacle | |
| CN112266881B (en) | Bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacle | |
| CN109355233B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN103642734B (en) | Microbacterium maritypicum and application thereof in preventing sugar beet disease-causing organisms | |
| US20220369648A1 (en) | Endophytic falciphora oryzae fo-r20 and its application | |
| CN101974438A (en) | Eucalyptus endophyte and application thereof | |
| CN108192838B (en) | Bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention | |
| CN109112069B (en) | Biocontrol endophytic fungus and application thereof | |
| US20220386625A1 (en) | Application of endophytic falciphora oryzae fo-r20 in controlling panicle blast | |
| CN111286477B (en) | Pseudomonas syringae and application thereof in prevention and treatment of apple ring rot | |
| CN110079484A (en) | A kind of bacillus subtilis and its application in agricultural production | |
| CN112342173A (en) | Bacillus belgii and application thereof | |
| CN113061551B (en) | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria | |
| CN112011478A (en) | Dendrobium nobile endogenous Burkholderia gladioli BL-HTie-5 and application thereof | |
| CN109112072B (en) | Application of trichoderma in promoting plant growth | |
| CN102851225B (en) | Stenotrophomonas acidaminiphila and application in control of apple tree canker thereof | |
| CN113234601B (en) | Novel marine fungus strain and application thereof in drought stress resistance of plants | |
| CN113736661A (en) | Screening method of salt-tolerant growth-promoting rhizobacteria, strain and application thereof | |
| CN106929433B (en) | Phosphate solubilizing penicillium and application thereof | |
| CN116790410B (en) | Bacillus cereus YZ-228 fermentation preparation and preparation method and application thereof | |
| CN116987615B (en) | Lecaniella albopictus YZ-151 and application thereof | |
| CN111139206A (en) | Growth-promoting compound endophytic bacteria agent and application thereof | |
| CN114990020B (en) | Biocontrol strain and application thereof | |
| CN116463220A (en) | Dark-color DSE fungus for promoting blueberry growth and application thereof | |
| CN111826290B (en) | Mangrove endophytic fungus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |